The lab of Ege Kavalali, Professor and Chair of the Department of Pharmacology, recently published groundbreaking research in Nature Communications that uncovers a critical role for liquid-liquid phase separation (LLPS) in the nanostructure of synapses. Their findings suggest that LLPS significantly influences synaptic function, particularly in the context of evoked neurotransmission. This new understanding of LLPS could reshape how we think about neuronal signaling and synaptic physiology.
The research explored the phenomenon of liquid-liquid phase separation within the neuronal cytoplasm. Given that the cytoplasm is predominantly aqueous, distinct liquid phases can spontaneously form within it. These phases are characterized by varying concentrations of macromolecules, which may include proteins or nucleic acids. The behavior of LLPS is similar to the interaction of oil and water—while both are liquids, they separate into different phases due to their differing physical properties. At the molecular level, this results in the formation of liquid condensates or membrane-less organelles. These liquid phases are enriched in certain protein-protein or protein-nucleic acid complexes, which help separate from a de-enriched phase. This dynamic compartmentalization within the cytosol allows cells to organize themselves in ways that are essential for cellular function, despite lacking traditional membrane-bound organelles.
The paper’s first author, Natalie J. Guzikowski, a Ph.D. student in the Kavalali lab, sat down to discuss the work and its significance. Here, she answers key questions about the research.
What Issue Does Your Research Address?
Over the last decade, research into LLPS has revealed its critical role in mediating protein interactions throughout the cell, including at synapses. However, one challenge that remained was understanding how these liquid condensates contribute to the physiology of neurons, particularly during synaptic signaling. The synapse is a highly complex and dynamic structure that does not have an enclosing membrane like other cellular organelles, such as the nucleus or mitochondria. Instead, it maintains its organization through non-membrane-bound mechanisms. Our research delves into how liquid-like properties contribute to synaptic function and help neurons effectively transmit signals across synapses, ensuring high-fidelity neurotransmission.
What Were Your Top Three Findings?
First, we found that the principles of liquid condensate biology are preserved within the nanostructure of synapses. This suggests that liquid-like properties help compartmentalize the synapse into distinct functional zones where different protein interactions occur in synchrony.
Second, we discovered that the organization of the presynaptic environment, including both synaptic vesicle pools and active zone scaffolding complexes, is fluid and dynamic in nature. These liquid condensates are essential for efficient and precise neurotransmission, emphasizing the importance of phase separation in regulating synaptic signaling.
Lastly, we demonstrated that LLPS is a necessary feature of the synaptic nanodomain. Disruption of this organization impacts evoked neurotransmission, but interestingly, spontaneous neurotransmission remains largely unaffected. This finding highlights the specificity of LLPS and its critical role in action potential-driven neurotransmitter release.
What Are Spontaneous and Evoked Neurotransmission?
Evoked neurotransmission refers to the release of neurotransmitters in response to an action potential. An action potential is an electrical signal that travels down the neuron, ultimately triggering neurotransmitter release at the synapse. This release then prompts an electrical signal in the next neuron, continuing the communication between nerve cells.
Spontaneous neurotransmission, on the other hand, occurs in the absence of an action potential. It is still an important process that underlies homeostatic plasticity, which helps maintain the stability of neural networks and allows the brain to adapt to changing conditions. Spontaneous neurotransmission also plays a role in the development of the nervous system and is involved in various psychiatric disorders. Though it occurs without the trigger of an action potential, spontaneous neurotransmission is fundamental to synaptic function and plasticity.
What Was Unique About Your Approach to the Research?
What set our research apart was our focus on manipulating LLPS complexes without targeting the typical pathways like protein domain interactions, genetic perturbations, or protein trafficking. This allowed us to establish the nanospecificity of LLPS at the synapse, revealing its physiological relevance without disrupting other cellular processes.
Moreover, our study used electrophysiology to monitor neurotransmission in real time, offering insights into how LLPS influences synaptic function at a level of specificity that had not been previously explored. Electrophysiological techniques allowed us to observe how disrupting LLPS affected neurotransmitter release, which provided a clear understanding of how LLPS impacts synaptic signaling in action.
What Are the Long-Term Benefits of This Research?
Our research could have far-reaching implications for understanding neurological diseases. By uncovering the specific role of LLPS in synaptic function, we provide insights into the molecular mechanisms underlying several neurological conditions. These include diseases related to synaptic dysfunction, such as Alzheimer’s disease, Parkinson’s disease, and autism spectrum disorders. Understanding the role of LLPS in neurotransmission could open new avenues for targeted therapies, potentially leading to the development of drugs that specifically modulate LLPS to restore proper synaptic function.
Additionally, our findings may contribute to the broader understanding of cellular organization in the brain. By further delineating how liquid condensates organize synaptic signaling, we could gain insight into how similar processes might occur in other complex cellular systems.
Where Is This Research Taking You Next?
This work is bridging the gap between what we know about LLPS in vitro—often in simplified systems—and how LLPS functions at the synapse in vivo. By continuing to study synaptic physiology at the intersection of structure and function, we hope to deepen our understanding of how phase separation influences not just neurotransmission but also neuroplasticity and brain function as a whole.
Our future research aims to explore how different synaptic proteins and complexes interact within LLPS condensates and how these interactions affect neuronal communication. We also plan to extend our studies to examine how disturbances in LLPS might contribute to diseases and how targeting these processes could provide new therapeutic strategies.
More information: Natalie J. Guzikowski et al, Functional specificity of liquid-liquid phase separation at the synapse, Nature Communications (2024). DOI: 10.1038/s41467-024-54423-7